NITROGEN-CONTAINING-COMPOUNDS AND SULFUR-CONTAINING-COMPOUNDS ENHANCETHE SYNTHESIS OF STORAGE RESERVES IN DEVELOPING SOMATIC EMBRYOS OF ALFALFA (MEDICAGO-SATIVA L.)

During alfalfa somatic embryogenesis, failure to synthesize the full c omplement of storage proteins (7S, 2S, 11S) has been a serious limitat ion in producing quality embryos. This study was directed at improving embryo quality through regulation of storage reserve synthesis, and t herefore culture media protocols were modified. B5h, B5g, and Boi2y me dia originally used for callus-induction, multiplication and embryo-in duction were replaced by SH medium. The effects on embryo-maturation o f glutamine, glutathione, and cystine (nitrogen- (N) and sulphur- (S) supplementation) in the callus-induction medium, and abscisic acid (AB A) in the embryo-induction medium were investigated. The total storage reserve content, especially storage proteins was used as the embryo q uality criterion. N- and S-supplementation in the callus-induction med ium had a positive effect on the amount and nature of callus produced per explant. However, the storage protein content of the embryo was no t enhanced. ABA supplementation in the embryo-induction medium, on the other hand, increased the total storage protein content (to about 29% of embryo dry weight), particularly in the high-salt soluble fraction . One dimensional SDS-PAGE of the low- and high-salt soluble fraction proteins showed that most of the major proteins were present equally. The improved somatic embryos accumulated starch equivalent to about 6% , and lipids equivalent to about 17-20%, of the embryo dry weight. Ele ctron microscopy studies showed that non-vacuolar starch and lipid acc umulation preceded vacuolar storage protein accumulation. Light microg raphs confirmed the improved storage protein status of the somatic emb ryos. (C) 1998 Elsevier Science Ireland Ltd. All rights reserved.